Article Figures & Data
Figures
- Figure 1. Plitidepsin shows strong antiviral activity in vitro against different coronavirus species.
(A) Treatment of Huh-7 cells with 0.5–50 nM of plitidepsin inhibited infection of a human coronavirus 229E expressing green fluorescent protein. All cells were treated 8 h after infection and fluorescent foci were analyzed at 48 h. (B) Accumulation of SARS-CoV genomic RNA is inhibited with increasing doses of plitidepsin. Confluent Vero E6 cells were infected with SARS-CoV and subsequently treated with plitidepsin at varying concentrations 1 hour post infection. Viral genomic RNA was measured 48 hours post infection. (C) Cytopathic effect on Vero E6 cells exposed to a fixed concentration of SARS-CoV-2 in the presence of increasing concentrations of plitidepsin. Plitidepsin was used at a concentration ranging from 5 nM to 100 μM. Nonlinear fit to a variable response curve from one representative experiment with two replicates is shown (blue), excluding data from drug concentrations with associated toxicity; cytotoxicity in the absence of virus is also shown (green). Error bars represent SD; points without error bars have a SD that is too small to visualize. DMSO, dimethyl sulfoxide; RLU, relative light unit.
- Figure 2. Plitidepsin shows strong antiviral activity in vitro against SARS-CoV-2 variants.
(A, B, C, D, E, F) Plitidepsin inhibits SARS-CoV-2 variants. HeLa-ACE2 cells were pretreated with plitidepsin or DMSO control 2 h after infection with (A) SARS-CoV-2/WA1, (B) α (B.1.1.7), (C) β (B.1.351), (D) δ (B.1.617.2), (E) μ (B.1.621), or (F) ο (B.1.1.529). Virus infectivity was measured 48 h postinfection. Cytotoxicity was performed in uninfected HeLa-ACE2 cells with same compound dilutions and concurrent with viral replication assay. Error bars represent SD across biologically independent triplicates. (G) Plitidepsin efficacy against early and α (B.1.1.7) variants compared to remdesivir. Calu-3 and Caco-2 cells were pre-treated with plitidepsin, remdesivir, or DMSO control at the indicated concentrations at an equivalent dilution for 2 h before SARS-CoV-2 infection. Cells were harvested after 24 h for analysis, and viral infection measured by intracellular detection of SARS-CoV-2 nucleoprotein by flow cytometry. Tetrazolium salt (MTT) assay was performed to verify cell viability. Error bars represent standard error of the mean. IC50: half maximal inhibitory concentration.
- Figure 3. Pharmacological estimation of active plasma concentrations of plitidepsin.
Predicted plasma concentrations achieved by a 90 min i.v. infusion of plitidepsin (1.5, 2, and 2.5 mg) and plasma IC50 and IC90 thresholds to assure concentrations in lung above IC50 and IC90 established in vitro, respectively (8, 52). Results were used to support the study doses and schedule. IC50: half maximal inhibitory concentration; IC90: 90% of maximal inhibitory concentration.
- Figure 4. Study Flow (CONSORT).
* For safety reasons, the first three patients of the study were sequentially allocated at the lowest dose level. Inclusion in the highest dose group was opened when three patients had been randomized to the intermediate dose. For that reason, the last three patients treated at the highest dose were also sequentially allocated. ** One patient withdrew consent before starting any study procedure and was replaced. *** All treated patients were evaluated for safety. All patients who completed treatment were assessed for efficacy. One patient experienced a grade 3 hypersensitivity reaction, shortly after the start of day 1 infusion of plitidepsin. This patient did not complete therapy, discontinued the study for safety reasons and was not evaluable for efficacy. This patient was not replaced. d, days; pts, patients.
- Figure 5. APLICOV-PC: Protocol Treatment and Pre-medication.
IV, intravenous; PO, oral; 5-HT3, serotonin (5-hydroxytryptamine) receptor 3.
- Figure S1. Intra-patient time-course variation in ALT, per dose-cohort.
Shadowed area represents treatment with plitidepsin on days 1 to 3. Each color line represents one patient. Bold line represents median values. ALT, alanine aminotransferase; ULN, upper limit of normal.
- Figure 6. APLICOV-PC Study: Preliminary Efficacy Outcomes.
(A) Viral load kinetics (qRT-PCR from nasopharyngeal exudates), by baseline severity of the disease (23). (B) Viral load kinetics (qRT-PCR from nasopharyngeal exudates), by dose of plitidepsin. LoQ: limit of quantification. See Table S4 for individual data results.
- Figure S2. Individual assessment of a six-category ordinal scale over time according to the severity of the disease at baseline.
The six-point scale was defined as follows (24): 1, discharged or having reached discharge criteria (defined as “clinical recovery”: normalization of pyrexia, respiratory rate <24 breaths per minute, saturation of peripheral oxygen >94% on room air, and relief of cough, all maintained for at least 72 h); 2, hospital admission but not requiring oxygen supplementation; 3, hospital admission for oxygen therapy (but not requiring high-flow or ventilation support); 4, hospital admission for noninvasive ventilation or high-flow oxygen therapy; 5, hospital admission for extracorporeal membrane oxygenation or invasive mechanical ventilation; 6, death.
- Figure 7. Post hoc analysis on hospital discharge by plitidepsin dose.
(A) Reverse Kaplan Meier plot showing the cumulative incidence of hospital discharge by plitidepsin dose. (B) Length of hospitalization, by plitidepsin dose and disease severity at baseline (23). Orange bars represent admission in intensive care units. Dashed lines labeled D8 and D15 are days 8 and 15, respectively, considering the start of therapy with plitidepsin as Day 1; this is equivalent to stays of 7 or 14 d from the start of therapy. See Figs S2S–SFigs S2–S4 for post hoc analysis on hospital discharge and respiratory support according to the severity of the disease at baseline.
- Figure S3. Post hoc analysis on hospital discharge, by baseline disease severity.
Reverse Kaplan Meier plot showing the cumulative incidence of hospital discharge by baseline severity (mild, moderate or severe COVID-19, according to FDA definition) (23).
- Figure S4. Post hoc analysis on hospital discharge, based on baseline disease severity and plitidepsin dose.
Length of hospitalization, by disease severity at baseline (23) and dose of plitidepsin. Orange bars represent admission in intensive care units. Dashed lines labeled D8 and D15 are days 8 and 15, respectively, considering the start of therapy with plitidepsin as Day 1; this is equivalent to stays of 7 or 14 d from the start of therapy.
- Figure S5. Intra-patient time-course variation in lymphocytes, per dose-cohort.
Shadowed area represents treatment with plitidepsin on days 1 to 3. Each color line represents one patient. Bold line represents median values.
- Figure S6. Intra-patient time-course variation in C-Reactive Protein, per dose-cohort.
Shadowed area represents treatment with plitidepsin on days 1 to 3. Each color line represents one patient. Bold line represents median values.
- Figure 8. Post hoc analysis on hospital discharge by plitidepsin dose in patients with moderate COVID-19 at baseline.
(A) Subgroup of patients with moderate COVID-19 (n = 23 pts): Distribution of the probability of the duration of the hospitalization, according to the dose of plitidepsin administered. (B) Subgroup of patients with moderate COVID-19 (n = 23 pts): Mean score over time of a six-category ordinal scale in patients with moderate disease at baseline, according to the administered dose of plitidepsin. The six-point scale was defined as follows (24): 1, discharged or having reached discharge criteria (defined as “clinical recovery”: normalization of pyrexia, respiratory rate <24 breaths per minute, saturation of peripheral oxygen >94% on room air, and relief of cough, all maintained for at least 72 h); 2, hospital admission but not requiring oxygen supplementation; 3, hospital admission for oxygen therapy (but not requiring high-flow or ventilation support); 4, hospital admission for noninvasive ventilation or high-flow oxygen therapy; 5, hospital admission for extracorporeal membrane oxygenation or invasive mechanical ventilation; 6, death. See also Table 3 and Figs S7 and S8.
- Figure S7. Inflammatory biomarkers in patients with moderate COVID-19 throughout follow-up.
(A, B, C, D) Infection and inflammation parameters in patients with moderate COVID-19 receiving plitidepsin, including (A) lymphocyte counts, (B) C-reactive protein, (C) neutrophil to lymphocyte ratio, and (D) D-dimers. Shadowed area represents treatment with plitidepsin on days 1 to 3. Mean and standard error of the mean are represented in the plots.
- Figure S8. Individual assessment of a six-category ordinal scale over time in patients with moderate COVID-19 at baseline, according to the administered dose of plitidepsin.
In patients with moderate COVID-19 at baseline (n = 23 pts), more patients treated at 2.5 mg/d were taken off supplementary oxygen (category 2) and were discharged from the hospital (category 1) by the end of the first week, with respect to other doses (non-statistically significant post hoc finding). The six-point scale was defined as follows (24): 1, discharged or having reached discharge criteria (defined as “clinical recovery”: normalization of pyrexia, respiratory rate <24 breaths per minute, saturation of peripheral oxygen >94% on room air, and relief of cough, all maintained for at least 72 h); 2, hospital admission but not requiring oxygen supplementation; 3, hospital admission for oxygen therapy (but not requiring high-flow or ventilation support); 4, hospital admission for noninvasive ventilation or high-flow oxygen therapy; 5, hospital admission for extracorporeal membrane oxygenation or invasive mechanical ventilation; 6, death. Categories 5 and 6 are not represented in the legend as there were no patients fulfilling the criteria. See also Fig 8B.
- Figure S9. Comparisons of radiological and inflammatory biomarkers in a 41-yr-old patient treated with 2.5 mg/day plitidepsin.
(A, B) Baseline CT scan of a participant subject, a 41 yr old male with moderate COVID-19 at baseline, (A) shows multiple bilateral lung infiltrates before plitidepsin treatment and (B) their status 7 d after treatment with plitidepsin 2.5 mg/day. (C) Changes to C-reactive protein and absolute lymphocyte count in the same patient.
- Figure S10. Lung alveoli from murine models.
Hematoxylin Eosin stained sections of lung from K18 hACE2 mice. (A, B, C) Lungs were harvested on day 3 postinfection (8): (A) lung tissue from a noninfected mouse, (B) alveolar inflammation in a SARS-CoV-2-infected mouse receiving vehicle, and (C) alveolar inflammation in a SARS-CoV-2 infected mouse receiving plitidepsin. Scale bar represents 100 μm.
Tables
Parameter 1.5 mg/daya (n = 15) 2.0 mg/daya (n = 15) 2.5 mg/daya (n = 15) Age (yr)b 51 (32–75) 49 (34–71) 53 (31–84) Gender—N (%) Male 11 (73.3%) 11 (73.3%) 8 (53.3%) Female 4 (26.7%) 4 (26.7%) 7 (46.7%) Race—N (%) White 13 (86.7%) 9 (60%) 9 (60%) Latino 2 (13.3%) 4 (26.7%) 6 (40%) Asian 0 (0%) 1 (6.7%) 0 (0%) Arab 0 (0%) 1 (6.7%) 0 (0%) Time from symptom onset to first administration (d)b 6 (3–10) 6 (3–10) 6 (2–10) Comorbidities—N (%) One 2 (13.3%) 7 (46.7%) 6 (40%) Two or more 8 (53.3%) 6 (40%) 7 (46.7%) Hypertension 2 (13.3%) 2 (13.3%) 5 (33.3%) Heart disease 1 (6.7%) 0 (0%) 1 (6.7%) COPDc 1 (6.7%) 1 (6.7%) 1 (6.7%) Asthma 2 (13.3%) 0 (0%) 3 (20%) Kidney disease 0 (0%) 1 (6.7%) 0 (0%) Diabetes 1 (6.7%) 5 (33.3%) 2 (13.3%) Obesity 1 (6.7%) 5 (33.3%) 4 (26.7%) Patients assessed at room air—N(%) 9 (60.0) 7 (46.7) 6 40%) SpO2 at room air (%)b 95 (92–99) 95 (91–97) 96.5 (94–97) PaO2/FiO2 ratiob 358 (336–408) 352 (285–396) 343 (253–481) PaO2/FiO2 ratio < 300 – N(%) 0 3 (20.0) 2 (13.3) Disease severity at entry—N (%) (1) Mild COVID-19 2 (13.3%) 3 (20%) 1 (6.7%) Moderate COVID-19 8 (53.3%) 7 (46.7%) 8 (53.3%) Severe COVID-19 5 (33%) 5 (33%) 6 (40%) D-dimer (ng/ml)b 330 (162–1,081) 463.5 (200–1,270) 415 (106–962) Ferritin (ng/ml)b 408 (96.8–1,652.8) 597 (174–1,055.2) 363 (12.2–1,647) C-reactive protein (mg/l)b 17.7 (1.2–120.4) 67.2 (2.1–128) 32.6 (0.3–120) log10 copies/ml viral load median (range)b 6.3 (1.5–9.7) 6.2 (3.8–7) 5.7 (1.5–10.6) Day 1 six-point ordinal scale—N (%)d (2) 2d 6 (40) 6 (40) 4 (26.7) 3d 9 (60) 9 (60) 11 (73.3) ↵a Daily for 3 consecutive days.
↵b Median (range).
↵c Chronic Obstructive Pulmonary Disease.
↵d The six-point scale was defined as follows (24): 1, discharged or having reached discharge criteria (defined as “clinical recovery”: normalization of pyrexia, respiratory rate <24 breaths per minute, saturation of peripheral oxygen >94% on room air, and relief of cough, all maintained for at least 72 h); 2, hospital admission but not requiring oxygen supplementation; 3, hospital admission for oxygen therapy (but not requiring high-flow or ventilation support); 4, hospital admission for noninvasive ventilation or high-flow oxygen therapy; 5, hospital admission for extracorporeal membrane oxygenation or invasive mechanical ventilation; 6, death.
PaO2, Oxygen partial pressure in arterial blood (imputed from oxygen saturation as described in Supplemental Data 2). FiO2. Oxygen proportion in inspired air.
Parameter Pre-amendmenta,b (n = 9) Post-amendmentc (n = 36) Grade 1 Grade 2 Grade 3 Grade 1 Grade 2 Grade 3 N (%) N (%) N (%) N (%) N (%) N (%) Nausea 3 (33.3%) 2 (22.2%) — 11 (30.6%) 3 (8.3%) — Vomiting 2 (22.2%) — — 3 (8.3%) 2 (5.6%) — Diarrhea — — — 1 (2.8%) 1 (2.8%) 1 (2.8%) Abdominal pain — — — 2 (5.6%) — — Dyspepsia — — — 2 (5.6%) — — Asthenia — — — 1 (2.8%) 1 (2.8%) — Anorexia — — — 1 (2.8%) — — Chest discomfort - - - 1 (2.8%) - - Temperature regulation disorder — — — 1 (2.8%) — — Dysthermia — — — 1 (2.8%) — — Anaphylactic reaction — — 1 (11.1%) — — — Amylase increasedd — — — — 1 (2.8%) — Lipase increasede — — — — 1 (2.8%) — Decreased appetite — — — — 1 (2.8%) — Dizziness — — — 2 (5.6%) — — Dysgeusia — — — 2 (5.6%) — — ↵a Relevant amendment #9 was implemented in Protocol v5.0 dated 13 August 2020 (Supplemental Data 2): It modified prophylactic medication before plitidepsin infusion to add ondansetron 8 mg IV slow infusion and changed the route of administration of dexamethasone, from oral to IV. The dose of dexamethasone was 8 mg (calculated as 8 mg dexamethasone phosphate, which is equivalent to 6.6 mg dexamethasone base).
↵b 25 plitidepsin IV infusions.
↵c 108 plitidepsin IV infusions.
↵d Short lasting, 5 min, retro sternal low intensity pain during first day IV infusion: self-resolved plitidepsin infusion completed days 1, 2, and 3.
↵e Same patient, onset day 2 plitidepsin, self-resolved in 48 h.
End point Dose cohort 1.5 mg (N = 14a) 2.0 mg (N = 15) 2.5 mg (N = 15) Total (N = 44) Mortality from Day 1 to Day 7 — — — — Day 15 — — — — Day 31b 1 (7.1) — 1 (6.7) 2 (4.5) Patients requiring invasive mechanical ventilation and/or intensive care unit admission Day 1 to Day 7 2 (14.3) 1 (6.7) 2 (13.3) 5 (11.4) Day 8 to Day 15 1 (7.1) 1 (6.7) 1 (6.7) 3 (6.8) Day 16 to Day 31 1 (7.1) 1 (6.7) 1 (6.7) 3 (6.8) Day 1 to Day 31 2 (14.3) 1 (6.7) 3 (20.0) 6 (13.6) Patients requiring noninvasive mechanical ventilation Day 1 to Day 7 4 (28.6) 0 1 (6.7) 5 (11.4) Day 8 to Day 15 3 (21.4) 0 2 (13.3) 5 (11.4) Day 16 to Day 31 1 (7.1) 1 (6.7) 1 (6.7) 3 (6.8) Day 1 to Day 31 5 (35.7) 1 (6.7) 2 (13.3) 8 (18.2) Patients requiring oxygen therapy at Day 7 12 (85.7) 12 (80.0) 11 (73.3) 35 (79.5) Day 15 4 (28.6) 1 (6.7) 4 (26.7) 9 (20.5) Day 31 0 2 (13.3) 1 (6.7) 3 (6.8) Day 1 to Day 31 12 (85.7) 12 (80.0) 11 (73.3) 35 (79.5) Mean change in viral load from baseline toc log10 copies/ml Day 4 −1.23 −1.49 −1.32 −1.35 Day 7 −2.55 −2.26 −2.25 −2.35 Day 15 −4.22 −2.70 −2.92 −3.25 Day 31 −4.70 −3.53 −3.49 −3.85 Mean time from baseline until undetectable viral loadc Days 11 14 14 13 ↵a One patient who experienced an anaphylactic reaction during the first plitidepsin infusion had treatment discontinued and was not considered evaluable for efficacy.
↵b One additional patient treated at 2.5 mg/day died on Day 57, because of COVID-19 complications.
↵c Results based on 42 patients at Day 4 (13 at 1.5 mg, 14 at 2.0 mg, 15 at 2.5 mg), 40 patients at Day 7 (13 at 1.5 mg, 14 at 2.0 mg, 13 at 2.5 mg), 38 patients at Day 15 (12 at 1.5 mg, 13 at 2.0 mg, 13 at 2.5 mg), and 39 patients at Day 31 (11 at 1.5 mg, 14 at 2.0 mg, 14 at 2.5 mg).
Supplemental Data 1.
Latest approved version of the protocol (v 7.0) with its relevant annexes.[LSA-2021-01200_Supplemental_Data_1.pdf]
Supplemental Data 2.
Summary of the protocol amendments and post hoc classifications and analyses.[LSA-2021-01200_Supplemental_Data_2.docx]
